WO1997000948A1

WO1997000948A1 - Oxidase, microorganisms producing the same and use of the same

Info

Publication number: WO1997000948A1
Application number: PCT/JP1996/001594
Authority: WO
Inventors: Takashi Echigo; Ritsuko Ohno; Shiro Fukuyama
Original assignee: Novo Nordisk A/S
Priority date: 1995-06-23
Filing date: 1996-06-12
Publication date: 1997-01-09
Also published as: AU1192797A; EP0852260A1

Abstract

A polyphenol oxidase which is produced by bacteria belonging to the genus Myrothecium and has the optimum pH value in the alkaline region of pH (8) or above; a process for producing the oxidase; Myrothecium verrucaria SD3001 (FERM P-14955) and Myrothecium roridum SD3002 (FERM P-15225) which are novel microorganisms producing the oxidase; and use of the oxidase in the fields of oxidizing, washing, etc. of colored materials and polyphenol-containing materials.

Description

TECHNICAL FIELD The present invention relates to a novel oxidase having an optimum reaction pH on the side of the enzyme and its use. More specifically, a novel polyphenol oxidase having an optimal reaction pH on the alkaline side of pH 8 or more produced by a microorganism, a microorganism producing the oxidase, a method for producing the oxidase, and coloring The present invention relates to a method for using the oxidase in fields such as oxidation treatment of substances and polyphenol-containing substances, and washing. Background art

Conventionally, polyphenol X-noroxidase and laccase produced by filamentous orchids such as basidiomycetes and incomplete fungi have been known as enzymes derived from microorganisms having a polyphenol oxidation activity. The conventional polyphenol oxidase has an optimum reaction pH from acidic to neutral range, and its activity is extremely low on the side of pH, especially on the side of pH 8 or higher. Actual applications were limited.

For example, the use of polyphenol oxidase for bleaching is described in W091-05839, EP91610032s DE4008894, JP-A-64-60693, and the like. However, washing operations such as washing are usually performed in the alkaline pH range, and particularly when oxidative bleaching is performed simultaneously in the presence of hydrogen peroxide, the pH is also increased to promote the bleaching action of hydrogen peroxide. It is desirable to perform the washing operation in Therefore, when using polyphenol oxidase for such purposes, It is practically difficult to use conventional enzymes that have an optimal pH for acidic pH.

Plant pigments such as flavonoids, xanthones and melanins and lignins are known as natural products having a polyphenol in the structural part, and polyphenol oxidase has an oxidizing effect on these natural products. In addition, polyphenol oxidase can be used as a reaction substrate for dichlorophenol and trichlorophenol, for which toxicity is a problem. Therefore, polyphenol oxidase is also useful in wastewater treatment containing these natural and non-natural products. However, conventional enzymes have an optimal reaction pH from acidic pH to neutral pH, so their use in the alkaline pH range is practically difficult, and this is a factor in the industrial use of polyphenol oxidase. This was a factor that narrowed the range of use.

On the other hand, among animal and plant-derived polyphenol oxidases, those having an optimum pH as high as pH 8 or higher are known (Comp. Biochem. Physiol., 1992, 102B, 4). , 891-896: Zhongguo Nongye Huaxue Huizhi, 1991, 29, 2, 177-185: Agric. Biol. Chem., 1991, 55, 1, 13-17). However, it is difficult to produce these polyphenol oxidases from tissues of animals and plants stably and inexpensively, and in order to use them for industrial use, the optimal reaction pH to the microorganism-derived alcohol is required. It has been desired to have a polyphenol oxidase. Disclosure of the invention

An object of the present invention is to search for a microorganism that produces porphyrenol oxidase having an optimum reaction pH on the side of pH 8 or higher, provide an oxidase produced by the microorganism, and provide an alkaline pH. To achieve a practical enzymatic oxidation of polyphenols in the region, and to utilize polyphenol oxidase It is to contribute to the expansion of the field. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a view showing a pH profile of polyphenoloxidase derived from the strain SD3001 of the present invention.

FIG. 2 is a diagram showing a temperature profile of polyphenoloxidase derived from strain SD3001.

FIG. 3 is a graph showing the temperature stability of polyphenoloxidase derived from strain SD3001.

FIG. 4 is a graph showing the pH stability of polyphenoloxidase derived from strain SD3001.

FIG. 5 is a diagram showing a pattern of the absorbance at 280 nm and the activity value of each fraction derived from the culture solution of the SD3001 strain.

FIG. 6 is a graph showing the absorbance of Fraction No. 59 derived from the culture of the SD 3001 strain.

FIG. 7 is a diagram showing the elution pattern of fraction No. 59 derived from the culture of the SD3001 strain in GFC analysis (HPLC).

FIG. 8 is a view showing a pH profile of a mixture of polyphenoloxidase derived from SD3001 strain and commercially available polyphenoloxidase.

FIG. 9 is a pH profile of polyphenoloxidase derived from the strain SD3002 of the present invention.

Figure 10 shows the temperature profile of polyphenoloxidase from strain SD3002.

FIG. 11 is a graph showing the temperature stability of polyphenol oxidase derived from SD 3002 strain. FIG. 12 is a graph showing pH stability of polyphenol oxidase derived from SD3002 strain.

FIG. 13 shows the absorbance at 280 nm and the activity value pattern of each fraction derived from the culture solution of SD 3002 strain.

FIG. 14 is a pH profile of a mixture of polyphenoloxidase derived from strain SD3002 and a commercially available polyphenoloxidase. Detailed description of the invention

The present inventors have enthusiastically searched for a wide range of microorganisms in search of microorganisms that produce an enzyme that catalyzes oxidation of a polyphenol substance in the pH range in the pH range. The search was extremely difficult, but finally a strain belonging to the genus Myrocesium (yrothecium), an incomplete fungus, produced the desired enzyme having an optimal reaction pH on the alkaline side with a pH of 8 or more. And completed the present invention.

That is, the present invention provides the following.

1) Polyphenol oxidase having an optimal reaction pH on the side of the microorganism having a pH of 8 or more.

2) The polyphenoloxidase according to 1), which is derived from a bacterium belonging to the genus Myrothecium.

3) The polyphenol oxidase according to the above 1) or 2), having the following properties.

(1) Action

Oxidizes polyphenols.

(2) Optimal reaction pH

PH 8.5 Has an optimum reaction ρΗ around 5-9.

(3) Optimal reaction temperature It has an optimal reaction temperature of about 6 crc.

(4) Molecular weight

The molecular weight measured by gel filtration chromatography analysis is about

62,000.

(5) Isoelectric point measured by isoelectric focusing is 4.5 ± 0.5.

4) The polyphenoloxidase according to 1) to 3), which can be obtained from Myrothecium verrucaria.

5) The polyphenoloxidase according to 1) to 3), which can be obtained from Myrothecium verrucaria SD3001 (accession number FEBM P-14955).

6) The polyphenol oxidase according to the above 1) or 2), having the following properties.

(1) Action

Oxidizes polyphenols.

(2) Optimal reaction pH

It has an optimal reaction pH around pH9.

(3) Optimal reaction temperature

It has an optimum reaction temperature around 70 ° C.

(4) Molecular weight

The molecular weight measured by gel filtration chromatography

60,000.

(5) The isoelectric point measured by isoelectric focusing is 4.1 ± 0.5.

7) The polyphenoloxidase according to the above 1), 2) or 6), which can be obtained from Myrothecium roridum. 8) The above 1), 2) or which can be obtained from Myrothecium roridum SD 3002 (Accession number FEP-15255) 6) The polyphenol oxiguse as described.

9) A phenol compound, an alkoxyl group-containing aromatic compound, a halogenated fininol compound, or an aromatic amine, wherein the polyphenol oxidase according to any one of 1) to 8) is acted on. Compound treatment method.

10) A method for treating a colored substance, which comprises applying the polyphenol oxidase according to any one of the above 1) to 8).

1 1) A method for treating a microorganism or a virus, which comprises causing the polyphenol oxidase according to any one of 1) to 8) to act thereon. 12) A method for treating paper, pulp, or fiber, wherein the polyphenol oxidase according to any one of 1) to 8) is acted on. 13) The use of the polyphenol oxidase according to any one of 1) to 8) above, which is used together with a detergent, a detergent, or a surfactant.

14) The method for using polyphenoloxidase according to any one of 1) to 8) above, which is used together with a substance having a peroxidase action.

15) Air, oxygen, ozone, hydrogen peroxide, hydrogen peroxide precursor, peracid precursor or peracid as an oxidizing agent alone or in combination of a plurality thereof, according to any one of the above 1) to 8) A method for using polyphenol oxidase, which is used together with polyphenol oxidase.

16) The method for using polyphenol oxidase according to any one of the above items 1) to 8), wherein the oxidase is used together with its substrate.

(17) The polyphenol according to any one of (1) to (8) above, wherein a bacterium belonging to the genus myrothecium is cultured. Method for producing oxidase.

18) The method for producing polyphenol-roxidase according to 17) above, wherein the bacterium belonging to the genus Myrothecium is Myrothecium verrucaria.

(19) The polyphenol according to (17), wherein the bacterium belonging to the genus Myrothecium is Myrothecium verrucaria SD 3001 (Accession number FEBM P-14955) or a mutant thereof. A method for producing a oxidase.

20) The method for producing polyphenol oxidase according to the above 17), wherein the fungus of the genus Myrothecium is Myrothecium roridum.

2 1) The polyphenol according to 17), wherein the fungus of the genus Myrothecium is Myrothecium roridum SD 3002 (Accession number FERM P-15255) or a mutant thereof. A method for producing Kishidase.

22) Myrothecium verrucaria SD 3001 (accession number FEM P-14955).

23) Yrothecium roridum Sd302 (accession number FERM P-15255).

24) A detergent composition comprising the polyphenoloxidase according to any one of 1) to 8).

[Production bacteria]

The strains belonging to the genus Myrothecium used for obtaining the polyphenoxylidase of the present invention include Myrothecium roridum, Myrothecium-vezoreca, Myrothecium verrucaria, and Myrothecium verrucaria. Two (Myrothecium prestonii), mirocesium ♦ Leuco Torikamu (Myrothecium leucotrichum) can be mentioned is a force, preferably a real opening cesium-Venoreka rear _(Myrothecium verrucaria) Λ Mi Roseshiumu • opening Ridamu (Myrot ecium roridum), particularly preferably Mi opening cesium-Vuerukaria SD 3 0 0 1 (Myrothecium verrucaria Use SD3001) or Myrothecium roridum SD3002.

On May 29, 1995, Myrothecium verrucaria SD3001 (Myrothecium verrucaria SD3001) was launched on May 1-3, 1995 at 1-3-1, Higashi, Tsukuba, Ibaraki, Japan. Deposited with the Institute as FERM P-14955 and transferred to the International Deposit on April 24, 1996 as FERM BP-5520. In addition, on October 26, 1995, Myrothecium roridum SD3002 (Myrothecium roridum SD3002) was researched by the Ministry of International Trade and Industry, National Institute of Industrial Science and Technology. Deposited as FEM P-15255 and transferred to the international deposit as FERM BP-5523 on April 24, 1996.

Observation of the color tone, shape and conidia, conidia formation structure, etc. of the colony on the maltoextract agar medium for a representative strain belonging to Myrothecium that produces the polyphenoloxydidase of the present invention The results of the observations made are shown below.

Item Property

Color of colony surface White to yellowish white, black conidia recognized Color of backside of colony White to yellowish white

Tissue on settlement surface Slightly wool-like

Mycelium colorless, smooth surface, width 1.5-3 / m

Filarid cylindrical, 3 to 6 rings

Size 10 ~ 15x 1.5 ~ 2 / D1

Conidium spindle type to lemon type

Size 6 ~ 8 X 2 ~ 3.5jum

-Part forms a fan-shaped accessory thread at the tip. This strain has a Cesium 《Venorecaria SD 3001 (Mvrothecium verrucaria SD3001), because some conidia form a fan-shaped accessory thread at the tip. Was named.

Another representative strain belonging to Mvrothecium which produces the polyphenoloxidase of the present invention was cultured on a potato carrot agar medium (PCA) at 25 ° C for 14 days. The results of morphological observations of the color, shape, conidium, and conidium formation structure of the village are shown below.

Item Property

Growth rate Village diameter 54-56 Sir

Colorless, smooth, 1.5 to 3 m wide

Filarid cylindrical, 3 to 6 rings

Size 12-16x 1.5-2.5t m

Conidia rod to thin oval, round at both ends but cut at base

Size 5.5 ~ 7.5 X 1.5 ~ 2 m

From the above observations, the present strain was named Mi port cesium Roridamu SD 30 02 (Myrothecium roridum SD3002) 0

[Preparation of enzyme]

The polyphenoloxidase of the present invention can be obtained by culturing the strain belonging to the genus Myrothecium and a mutant thereof, and can also be prepared using genetically engineered bacteria. That is, together with a DNA sequence encoding the polyphenol oxidase, an appropriate promoter having an enzyme expression function in the host organism, an operator, and a DNA promoter, the DNA sequence is combined with the DNA sequence in the host organism. A host cell transformed with an expression vector inserted into a DNA vector having an origin of replication for replicating the vector at the same time, or a DNA sequence encoding the polyphenol oxidase and a host organism. The host cell transformed by integrating the host cell DNA with a suitable promoter having an enzyme expression function, an operator, a terminator and a DNA sequence under the conditions capable of expressing polyphenoloxidase. It is also produced by a method of culturing the cells and recovering polyphenol oxidase from the medium.

0 In addition, the polyphenol oxidase of the present invention has a conventional optimal reaction on the acidic side based on the knowledge about the amino acid sequence of polyphenol oxidase obtained based on the DNA sequence encoding the polyphenol oxidase. It may be produced by a protein engineering method that modifies the DNA of polyphenoloxidase having PH.

In order to obtain a DNA fragment encoding the polyphenoloxidase of the present invention, for example, cDNA or a genomic library from the strain of the present invention is used as an isolation source, and the amino acid sequence of the polyphenoloxidase of the present invention or Identify the target DNA fragment using oligonucleotides synthesized based on the amino acid sequence of a known polyphenoloxidase as a probe, select a clone that expresses enzymatic activity, or use an antibody against the polyphenoloxidase Selection of a clone that produces a protein that reacts with E. coli can be performed by a conventional method.

The culture for obtaining the polyphenoloxidase of the present invention can use a commonly used synthetic medium or a nutrient medium containing an organic carbon source and an organic nitrogen source. Also, it is desirable to add Cu ²⁺ ion as a metal salt at a concentration of 0.001 mM to 1 OmM, preferably O. OlmM to 1 mM. The culture temperature is from 10 to 35 ° C, preferably from 20 to 30 ° C. An appropriate cultivation time is 20 hours to 150 hours, preferably 40 hours to 100 hours. The secreted polyphenoloxidase can be recovered from the medium by a known method. This collection procedure can be performed by a series of procedures in which cells are separated from the medium by centrifugation, filtration, or membrane separation, and chromatography is performed by, for example, ion exchange chromatography. Also, membrane concentration using an ultrafiltration membrane is effective.

[Properties of enzymes] (1) Enzyme from myrocesium-venorecalia (CMyrothecium verrucaria ^) SD3001

The enzyme derived from Myrothecium verrucaria SD 3001, which is a representative example of the polyphenol oxidase of the present invention, has a molecular weight of about 62,000 as measured by gel filtration and mouth chromatography, and is isoelectric focusing. The isoelectric point measured by is 4.5 ± 0.5.

This enzyme can perform oxidation reaction of polyphenols over a wide pH range of 5 to 11, but the preferred reaction pH is 10 to 10, and the more preferable reaction pH is 8 to 9.5. The optimum pH is around 8.5 to 9, which has the advantage of catalyzing the oxidation reaction on the alkaline side (Fig. 1), and the optimum temperature is about 60 ° C (Fig. 2). Shows that the activity after heat treatment for 30 minutes at various constant temperatures is, for example, about 100% residual activity at 60 (Fig. 3). The residual activity after treatment at 30 ° C for 30 minutes in the phage is stable over a wide range of pH (Fig. 4) .These results indicate that CMrothecium verrucaria ) Oxidation reaction in various medium and low temperature solutions over a wide pH range I guarantee that.

(2) Enzyme derived from Myrothecium roridum SD3002

The enzyme derived from Myrot_hecium roridum SD3002, which is a representative example of the other polyphenol oxidases of the present invention, has a molecular weight of about 60, determined by gel filtration chromatography analysis. 000, and the isoelectric point measured by isoelectric focusing is 4.1 ± 0.5.

This enzyme can oxidize polyphenols in a wide pH range of 6 to 12, but the preferred reaction pH is 8 to 11, and the more preferred reaction pH is 8.5 to

Two It has an optimum pH near pH 9 and catalyzes the oxidation reaction on the alkaline side (Fig. 9). The optimum temperature for a reaction at pH 8.7 for 10 minutes is around 70 ° C (Fig. 10). Furthermore, the activity after heat treatment at 70 ° C. for various times shows, for example, approximately 50% residual activity when treated for 30 minutes (FIG. 11). In addition, the residual activity after treatment at 30 ° C. for 30 minutes in a buffer having various pH values shows stability over a wide range of pH values as shown in FIG. These results confirm that the enzyme from Myrothecium roridum SD 3002 can oxidize in a wide range of neutral to alkaline pH ranges in a variety of medium and low temperature solutions. Is what you do.

The poly (X-noroxidase) of the present invention can be used in combination with a conventional enzyme having an optimal reaction pH on the acidic side. In other words, by using a conventionally known polyphenol oxidase having an optimum reaction pH on the acidic side in combination with the polyphenol oxidase of the present invention, the polyphenol oxidase reaction can be performed in a wide pH range from acidic to alkaline. It is possible to do. When enzymes are mixed and used for such a purpose, the mixing ratio between the activity of the polyphenol oxidase having the optimum reaction PH on the acidic side and the activity of the polyphenol oxidase of the present invention is preferably 1 L0: 1, more preferably 1: 3 to 3: 1. The polyphenol oxidase of the present invention is also useful in that it allows a polyphenol oxidase reaction to be achieved in a wide pH range.

[Enzyme activity measurement method]

In the present invention, the measurement of the polyphenol oxidation activity of polyoxidase includes syringaldazine and lO OmM Tris-HC1 buffer solution (pH 8.7) at 20 ° C. and 20 ppm. water

Three Perform the reaction in a solution and measure the absorbance at 525 nm. The activity of oxidizing 1〃mo 1 of syringaldazine per minute was defined as 1 unit (hereinafter abbreviated as U).

[Use]

As an application of the novel polyphenol oxidase of the present invention having an optimal reaction zone to pH, for example, application to bleaching is possible.

The use of polyphenol oxidase for bleaching is described in WO91-05839, DE4008894, JP-A-64-60693, and the like. Washing operations such as washing are usually performed at an alkaline pH, and especially peroxidation. When oxidative bleaching in the presence of hydrogen is performed at the same time, a washing operation with alkali pH is desirable to promote the bleaching action of hydrogen peroxide. Conventional polyphenol oxidase, which has an optimum pH for acidic pH and an optimal pH for acidic pH, has been difficult to practically utilize for such uses. The polyphenol oxidase of the present invention is useful for opening the use of polyphenol oxidase in the field of washing and bleaching of alcohol.

Oxidative bleaching with hydrogen peroxide is now widely used in washing and laundering. However, hydrogen peroxide does not have sufficient bleaching power at low temperatures below 60 ° C. To improve this, a peracid precursor is used together with hydrogen peroxide, but the bleaching power at a low temperature of 40 ° C or lower is not sufficient, and a more effective bleaching system is required. Therefore, various enzymatic bleaching acceleration methods have been proposed. The oxidative bleaching of the alkaline PH by coexisting the polyphenolase according to the present invention with one or more of the substances having a peroxidase action such as peroxidase, lignin peroxidase, manganese peroxide or the like. The usefulness of the polyphenol oxidase of the present invention is clear. Hydrogen peroxide, which is widely used in oxidative bleaching, is an expensive oxidizing agent, and hydrogen peroxide precursors, peracid precursors, and peracids, which are often used in cleaning agents, are more expensive oxidizing agents. It is. In addition, it is possible to generate hydrogen peroxide enzymatically by using oxidase and its substrate, but such a hydrogen peroxide generating system can also be considered as an expensive oxidizing agent. The polyphenol oxidase of the present invention can be used to convert air, oxygen, ozone, hydrogen peroxide, hydrogen peroxide precursor, peroxy acid precursor, or peroxide, which is an oxidizing agent conventionally used for oxidative bleaching. Oxidative bleaching can be promoted by using an acid alone or in combination. Therefore, the usefulness of the polyphenol oxidase of the present invention which can achieve oxidative bleaching by effectively using an oxidizing agent is clear.

Among the oxidizing agents, the hydrogen peroxide precursor dissolves in water to form perhydroxylion. Such substances include monohydrate or tetrahydrate perborates, percarbonates, perborates, sodium perpyrophosphate, perbenzoic acid, urea hydrogen peroxide There are a reactant, a melamine-hydrogen peroxide reactant, and a citrate perhydrate, and perborate and percarbonate are particularly preferable. Furthermore, a hydrogen peroxide generation system using oxidase and its substrate can be used as the hydrogen peroxide precursor. Examples of such oxidases are glucose oxidase, alcohol oxidase, glycerol oxidase, amino oxidase, amino acid oxidase, D-amino acid oxidase, aryl alcohol oxidase, aldehyde oxidase, and galactose. Examples thereof include soxidase, sorbose oxidase, uretoxidase, xanthine oxidase, and cholesterol oxidase, and particularly preferred are glucose oxidase and alcohol oxidase. Further, the peracid precursor is an organic compound having a reactive acyl group or

Five These include boric acid esters, carboxylic anhydrides, and acetates. Such substances include TAE D (tetra acetyl ethylene Qiamine), _N AMD (tetra acetyl methylene diamine) TAGU (tetra acetyl glycoluril) DADHT (diacetyl dioxohexahydro triazine) S NO BS (sodium nonanoy 1 oxybenz ene sulfonate) IS 0 N 0 BS (sodium isononan oyloxybenzene sulfonate) oxalic anhydride, benzoic anhydride, phthalanoic anhydride, PAG (glucose pentaacetate), xylose tracetate And TAED S NO BS is particularly preferred.

Furthermore, examples of the peracid include D PDDA (diperoxydodecanedioic acid) DPIPA (diperoxyisophthalic acid) MMP PH (magnesium monoperoxyphthalate hexahydrate) NAPAA (nonylamidop eroxyadipic acid) ₀

The polyphenoloxidase of the present invention can be used with various detergents, detergents or surfactants. As a result, a detergent or detergent composition containing the polyphenol oxidase of the present invention is provided. Representative examples of such detergents or detergent compositions are: 150% by weight surfactant, 0.50% by weight builder, 150% by weight alkaline agent per weight of detergent or detergent composition. Alternatively, a detergent or detergent composition comprising at least one compound selected from the group consisting of an inorganic electrolyte, 0.110% by weight of a redeposition inhibitor, an enzyme, a bleaching agent, a fluorescent dye, a caking inhibitor and an antioxidant. Things.

As the surfactant, for example, a linear or branched alkyl or alkenyl sulfate, an amide sulfate, a linear or branched alkyl or alkenyl group, ethylene oxide, propylene oxide and Aliphatic sulphates such as alkyl or alkenyl tersulphate to which single or multiple components of butylene oxide are added, alkyls

6 Aliphatic sulfonates such as sulfonate, amide sulfonate, dialkyl sulfosuccinate, α-olefin, vinylidene type and internal sulfonic acid, linear or branched alkylbenzene Aromatic sulfonic acid salts such as sulfonic acid salts, linear or branched alkyl or alkenyl groups, and one or more of ethylene oxide, propylene oxide and butylene oxide added Alkyl or alkenyl ether carboxylates or amides, monosulfo fatty acid salts or esters, amino acid surfactants, phosphates such as alkyl or alkenyl acid phosphates, alkyl or alkenyl phosphates Surfactant, sulfonic acid type amphoteric surfactant, solid Amphoteric surfactants, linear or branched alkyl or alkenyl groups, and alkyl or alkenyl to which one or more of ethylene oxide, propylene oxide and butylene oxide are added Polyoxetylene alkyl phenyl ether containing ether or alcohol, linear or branched alkyl or alkenyl group, and one or more of ethylene oxide, propylene oxide and butylene oxide added Detergent compositions such as higher fatty acid alkanolamides or alkylene oxide adducts thereof, sucrose fatty acid esters, fatty acid glycerin monoesters, alkyl or alkenyl amine oxides, and tetraalkylammonium salt type cationic surfactants By any of the surfactants are usually formulated as also available. In the case of an anionic surfactant, the counter ion is preferably a sodium ion or a potassium ion. These surfactants are used alone or as a mixture of two or more.

Orthophosphates, pyrrolinates, tripolyphosphates, methacrylates, hexamethacrylates, phthalates and builder agents and inorganic electrolytes

7 Phosphates such as citrate, ethane-1,1-diphosphonic acid and its derivatives, ethanehydroxy-1,1,2-triphosphonic acid, ethane-1,2-dicarboxy-1,2, -diphosphonic acid, methanehydric acid Phosphonates such as droxyphosphonic acid, 2-phosphonobutane-1,2-dicarboxylic acid, 1-phosphonobutane-1,2,3,4-tricarboxylic acid, phosphonocarboxylates such as α-methylphosphonosuccinic acid, aspartic acid, Amino acid salts such as glutamate, triamino triacetate, amino diacetate such as ethylene diamine tetraacetate, diethylene triamine pentaacetate, polyacrylic acid, poly itaconic acid, poly maleic acid, and maleic anhydride Polymer electrolytes such as acid copolymers, carboxymethylcellulose salts, polyethylene glycol, polyvinyl alcohol Non-dissociated polymers such as alcohol, carboxymethylated products such as diglycolic acid, oxysuccinic acid, carboxymethyloxysuccinic acid, dalconic acid, cunic acid, lactic acid, tartaric acid, sucrose, lactose, and carboxy of pentaerythritol Methylated compounds, carboxymethylated dalconic acid, organic acid salts such as benzenepolycarboxylic acid, oxalic acid, linoleic acid, oxydisuccinic acid, and dalconic acid; aluminosilicates such as zeolite; carbonates; Inorganic salts such as sulfates and metasilicates can be used as alkali metal salts.Organic substances such as starch and urea and inorganic compounds such as sodium chloride and bentonite can be used. Triethanolamine, ethanol Emissions, monoethanolamine Amin, etc. triisopropanolamine Amin can be used.

As described above, the detergent composition of the present invention contains a surfactant, the polyphenol oxidase of the present invention, and the like as constituent components, and if necessary, an amphoteric surfactant, such as perborate and carbonate. Bleach, pigment, builder, such as polyethylene glycol, polyvinyl alcohol

8 Anti-contaminating agents such as water, polyvinylpyrrolidone and carboxymethylcellulose, anti-caking agents and antioxidants, for example, other enzymes such as other oxidases and beloxidases, proteases, lipases, amylases and cellulases. be able to.

Any method may be used to incorporate the enzyme such as polyfuno-loxidase of the present invention into the detergent composition.However, the incorporation of the enzyme in the form of a fine powder is only possible when the detergent is used due to dust generation when handling the detergent. It is not preferable for the safety and health of workers in the detergent industry and the detergent industry, but it is preferable to shape it in a solution state or a shape in which dust generation is suppressed in advance.

This shaping may be performed by any of commonly used malm granulation, extrusion granulation, fluidized granulation, centrifugal fluidized granulation and other methods, but the enzyme to be added to the detergent composition of the present invention. The shape is not particularly limited to those formed by these methods.

For the purpose of delignification and bleaching, in a part of the pulp process, the bacterial strain of the present invention is inoculated to produce the polyphenol oxidase of the present invention, or the enzyme preparation of the present invention is directly added to the chip. Biopulping and biobleaching applied to ground pulp and the like are mentioned as useful application fields. Also in this case, the polyphenol oxidase of the present invention having the optimum reaction pH on the alkaline side is more suitable for adjusting the pH than the conventional polyfuninol oxidase having the optimum reaction pH on the acidic side. The cost can be reduced because the amount of chemical used in the process can be reduced.

Plant pigments such as flavonoids, xanthones, and melanins and lignins are known as natural products having a polyphenol in the structural part, and polyphenol-oxidase has an oxidizing effect on these natural products. Polyphenol oxidase also inhibits AOX (organic base) such as dichlorophenol and trichlorophenol, for which toxicity is a problem. Can be used as a substrate. Therefore, for example, the polyphenol oxidase of the present invention is useful also in wastewater treatment containing these natural products and non-natural products 4) o

Also, the biosensor using the polyphenoloxidase of the present invention monitors aromatic compounds in various aqueous solutions and organic solvents having a neutral to alkaline pH, reflecting the characteristics of the present enzyme. It can be used for

It is also possible to efficiently kill and inactivate microorganisms and viruses in the neutral to alkaline pH range by utilizing the reactivity of phenol radicals generated by the polyphenol oxidase of the present invention. It is possible. In other words, in addition to the bactericidal properties of the polyphenoloxidase substrate itself, it is possible to provide stronger bactericidal properties by enzymatically generated phenoxy radicals. In addition, if the sterilized product is subsequently contacted or ingested by the human body or released into the environment, the substrate for polyphenol oxidase is converted to a substance with reduced toxicity by oxidation, so it is necessary. It is useful because it can achieve both bactericidal properties at a particular point in time and subsequent safety.

In addition, when a polymer is synthesized using phenoxyradicalquinones generated by polyphenoloxidase, the applicable reaction can be expected to be expanded, and the polyphenoloxidase of the present invention is useful.

Furthermore, when oxidizing a group of substances containing oxidizable polyphenols such as catechols, an aliquot capable of simultaneously advancing the autooxidation reaction at pH and enzyme-catalyzed oxidation reaction of polyphenols. The use of polyphenoloxidase at pH is extremely effective for efficient oxidation. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, typical examples of the present invention will be shown and will be described more specifically. However, these are merely examples, and the present invention is not limited to these. Example 1: Culture and concentration of Myrothecium verrucaria SD3001 strain

A two-liter flask was used as a culture device, and 0.5% glucose and 0.1% (NH ₄ ) ₂ SO _{/ |} . 1.34% Na HPO _| · 12 H ₂ 0.3% KH ₂ P〇 ₄ , 0.1% N a C 0.1% peptone, 0.01% yeast extract, to those with 8 to p H by adding 2 NN a OH to the medium of 400 ml containing 0.1 mM C u S 0 _4, Mi Roseshiumu * Vuwerukaria (Myrothecium verrucaria) SD 3.001 (Accession No. FERM P-14955) was inoculated, and shaking culture was carried out at 27 ° C for 3 hours. After the culture, the culture broth was removed by centrifugation at 4 ° C. This was further concentrated and purified by an ultrafiltration membrane to obtain a crude purified concentrated aqueous solution (20 U / m 1) purified and concentrated to a fractionation range of 10,000 to 100,000. Example 2: Crude purification

_{1.34% N a 0 H PO ^} · 12 H 2 0, 0.3% KH 2 P 0 4, were equilibrated with 0.1% N a C 1 DEAE- C ellulofine A- 8 00m ( Seikagaku Corporation) column ( The culture broth after the eradication described in Example 1 was applied to the space above the column on the top of (¾620 mm, 50 cc), and the collection of the eluate was started in one fraction of 15 ml at the same time as the start of the passage. After applying 345 ml of culture broth, wash the column with a buffer of the same composition as used for equilibration, and increase the NaC1 concentration to 0.15M.

Two Elution was performed stepwise. Fig. 5 shows the absorbance at 280 nm and the pattern of polyphenol oxidase activity of each fraction. Of the fractions, No. 1 to 55 eluted with 0.1% NaC1, while No. 56 to 75 eluted with 0.15 MNaC1. Enzyme activity was strongly detected in fractions N 0.59 and 60. Table 1 shows the details of fraction Nos. 59 and 60.

Fraction Activity at 280 nm Purification magnification

No. Absorbance (unit / ml)

59 3.32 32.4 1 12

60 4.17 15.1 42 As a result of measuring the absorbance of fraction No. 59 in the range of 380 nm to 700 nm, an absorption maximum was observed at around 600 nm (Fig. 6). O Example 3: Substrate specificity

Substrate specificity of the polyphenol compound oxidation reaction was examined using the crude polyphenol oxidase of fraction No. 59 described in Example 2. At room temperature (20 ° C), measurement was performed by measuring the difference in oxygen consumption rate between 0.05 mM substrate and lO OmM Tris-HC1 buffer solution (pH 8.7) with and without enzyme addition. . Table 2 shows the results. Table 2 Substrate oxidation reaction

4—anisidine + + +

0—Ferrendiamine + + +

Silingardazine + + +

Syringic acid + + +

Ferulic acid ++ Example 4: Molecular weight

The molecular weight was measured using GFC (gel filtration chromatography). _{That, 1.34% N a 2 HP 0} 4 · 1 2 H 2 0, 0.3% KH 2 P 0 4, l% N a C l GFC column equilibrated with O connexion flow rate 1.0 m 1 / min to (Shodex PROTEIN KW The analysis, fractionation and activity measurement of crude polyphenol oxidase of fraction No. 59 described in Example 2 were carried out by HPLC using -802.5, duplicate and UV detector (280 nm). At this time, the polyphenoloxidase activity peak had a molecular weight of 62,000. The molecular weight marker-protein was MW-Marker (HPLC) manufactured by Oriental Industries, Ltd. The elution pattern of fraction No. 59 in the above GFC analysis (HPLC) is shown in FIG. Example 5: Bleaching treatment of black tea-contaminated cloth

50 mM sodium carbonate buffer containing 0.1% Tween 80, 0.02% activated zeolite powder (pH 9.0) Further, here, the partially purified polyphenol oxidase of fraction No. 59 described in Example 2 was combined with another bleaching agent in advance. The mixture thus prepared was added, and the mixture was shaken at 30 ° C. for 40 minutes for washing and bleaching. After that, it is washed with water and air-dried, and the Y, y, and X values based on the XY Z color system (CIE (International Commission on Illumination) standard color system) are measured using a colorimeter (CR-200, manufactured by MINOLTA). formula

Z = (1-X-y) Y / y

To calculate the Z value. Then, the increase in whiteness was evaluated based on the difference (ΔΖ value) between the Z values before and after the treatment. Table 3 shows the results.

For the tea-contaminated cloth, add 160 g of commercially available black tea (Hotel Restaurant Tran Ceylon, Japan Black Tea Co., Ltd.) to 5 liters of boiling water, boil for 5 minutes, remove the black tea by filtration, The filtrate was boiled for 5 minutes, put a cotton white cloth, boiled again for 5 minutes, removed the cloth, dried, washed thoroughly with ion-exchanged water and dried again. Table 3 Additives △ z value No additives 0.2 l UZral crude enzyme solution

200ρρπ! Hydrogen peroxide 4, 8

1 UZml crude enzyme solution + 200 ppm hydrogen peroxide 6.5

200ρρπι ノヽ. One strength-Bonnet · Na salt 2.8

1 UZinl crude enzyme solution + 200ρρπι Parker carbonateNa salt 6

200ρρπι Parker carbonate '' Na salt + 50ppm TAED 6.1

1 U / ral crude enzyme solution + 200 ppra carbonate * Na salt + 50 ppra TAED 8.1

200ppra Carbonate 'Na salt + 50ppni SNOBS 7.2

1 U / ral crude enzyme solution + 200ρρπ power-bone * Na salt + 50ppm SNOBS 8.7 Example 6: Cultivation and concentration in a 5-liter culture tank, and crude purification

0.5% glucose and _{0.1% N a N0 3, 1.34} % N a 2 HP 0. · 1 2 H 2 0, 0.3% KU 2 P_〇 _{4, 0. l% N a C} l, 0.2% peptone, 2 0 ppm yeast _{extract, 0.01% Mg SO 4 * 7} H 2 O, 5 Li, including those with a p H 8 by addition of 0.1 mM C u S 0 1 0 % to 3 medium re Tsuta consisting ₄ N a 0 H A single cultivation tank was inoculated with Myrothecium verrucaria SD 3001 (accession number FEP-14955) and shake-cultured at 28 ° C for 3 days. After the culture, 2.5 liters of a culture broth that had been orchid-removed was obtained by centrifugation at 4 ° C.

Next, a portion of this culture broth was purified by a minitan ultrafiltration system (Millipore) using a minitan filter bucket (CAT. NO .: PTGC0MP04, manufactured by Millipore) with a molecular weight of 10%. It was concentrated as more than, 000 fractions. This was further subjected to DE AE-Cellulofine A-800m column chromatography, and the eluted active fraction was again concentrated by the above-mentioned ultrafiltration system as a fraction having a molecular weight of 10,000 or more. Shrunk. Furthermore permeability after crystallization the reconcentrated liquid to _{_{2 OO p pmNH 4 HC Ο π}} , subjected to freeze-drying to obtain a crude product as a freeze-dried product. The lyophilized product had a polyphenoloxidase activity of 10 U Zmg. Example 7: Treatment of stained cloth with enzyme-containing detergent

25% by weight of sodium linear alkylbenzene sulfonate (LAS) and 5% by weight of polyoxyethylene lauryl ether, 15% by weight of sodium triphosphate, 6% by weight of sodium gayate, 1% by weight force Lupo carboxymethylcellulose Na Application Benefits um, the index detergent 1 0 gram of 4 8% by weight of N a ₂ S 0 _4, a material obtained by 0.1 g added pressure to the freeze-dried product described in example 6 as an enzyme formulated detergent Yeasts without added freeze-dried products An unmixed detergent was used.

A contaminated cloth was prepared by adding 100 ml of Evans Pro (Wako Pure Chemical Industries, Ltd.) 0.2 ml to the center of a cotton white cloth (5 cm x 5 cm). Next, after adding 1 piece of contaminated cloth and 1 Om1 of water to a 500 m1 beaker, add 1 Omg of detergent containing enzyme or detergent without enzyme, and shake for 12 minutes to wash. Was done. The stained cloth after treatment was washed with water and air-dried, and the Z value was calculated using a colorimeter (CR-200, manufactured by MINOLTA) in the same manner as in Example 5. A 6-point improvement in whiteness was shown. Example 8: Bleaching of crude pulp

The 5 0 0 ml beaker one volume used for culturing apparatus, 0.3% glucose and _{0.05% (NH ^) 2 S} 0 4, 1.34% N a Ri HP 0 ₄ · 1 2 H. _{_{0, 0.3% KH 2 0 4}} . 0.1% N a C 0.02% peptone, 50 p pm yeast E key _{scan, 0.02mM C u S 0 4,} 8% hardwood unbleached kraft pulp (L UK P) (Kappa one The medium was adjusted to pH 8.5 by adding 2 N NaOH to a 5 Om 1 medium containing a bacterium of 18) and inoculated with Myrothecium verrucaria SD 3001 (Accession number FERM P-14955). After the stationary culture at 4 ° C. for 4 days, the titer value was measured. Example 9: Polyphenol oxidase (from Takara Co., Ltd.) commercially available as a mixed reagent of an enzyme derived from Mv_rothecium verrucaria SD 3001 and a conventional polyphenol oxidase 0.1 U / m 1 solution and a 0.2 UZm 1 solution of the polyenoloxidase of the present invention mixed in equal volumes were used. When the activity was measured at various pH values, the oxidation reaction proceeded in a wide range of pH from 5 to 11, as shown in FIG. Example 10: Culture and concentration of Myrothecium roridum SD3002 strain

With 2 Li Tsu evening flask Ichiyo the culture device, 1% glucose and 0.2 ^ NH ^ C 1, 1.34 % N a 2 HP 0 ^ · 12 H 2 0, 0.3% KH. _{P 0 t, 0.1% N a} C 0.2% peptone, 0.05% Mg S_〇 ₄ · 7H ₂ 0, and 0.02mMC u S 0 ₄ 8 is added to bring the pH 2 N-N a OH to the medium of 550 ml comprising 27, and inoculated with Myrothecium roridum SD 3002 (Accession No. FERM P-15255). C, shaking culture for 90 hours was performed. After the culture, the culture broth was removed by centrifugation at 4 ° C. This was further concentrated and purified by an ultrafiltration membrane to obtain a partially purified concentrated aqueous solution (0.4 U / m 1) purified and concentrated to a fractionation range of molecular weight of 10,000 to 100,000. Example 11: Crude purification

_{_{1.34% N a 0 HP 0 4}} · 12 H 2 ◦, 0.3% KH 2 P_〇 _4, 0.1% N a C equilibrated with 1 DEAE- C e 1 1 u 1 ofine A- 800 m ( Seikagaku ( The culture broth after disinfection obtained in the same manner as described in Example 10 was applied to the top of the column (026 mm, 90 cc) in the space above the column. Recovery of the eluate started at 16 ml. After applying 480 ml of culture broth, elution was carried out by force ram washing with a buffer having the same composition as that used for the equilibration, and stepwise increasing the NaC1 concentration to 0.12M. The absorbance at 280 nm and the activity of polyphenol-oxidase activity for each fraction The turns are shown in Figure 13. Among the fractions, No. 1 to 70 were eluted with 0.1% NaC1, and N 0.71 to 10 (elution was performed with Hi0.12M NaC1. Enzyme activity Was strongly detected in fractions 80 to 84. Example 12: Substrate specificity

Using the partially purified polyphenol oxidase of Fraction No. 82 described in Example 11, the substrate specificity of the polyphenol compound oxidation reaction was examined. At room temperature (20 ° C), this was done by measuring the difference in oxygen consumption rate between 0.05 mM substrate and 10 OmM Tris-HCl buffer solution (pH 8.7) with and without enzyme addition. . Table 4 shows the results. Table 4 Oxidation reaction

Syringardazin + +

4 Anissidine +

o—Fenirangian +

Fururic acid Example 13: Molecular weight

The molecular weight was measured using GFC (gel filtration chromatography). That is, 1.34% Na ₂ HPO, 12H _O O, 0.3% KH. P

Two

The GFC column (Shodex PROTEIN-802.5, 2 columns) which had been flattened at a flow rate of 1.0 m 1 / min with 0% and 1% NaC 1 and HPLC using a UV detector (280 nm) was used. Of fraction No. 82 Analysis, fractionation and activity measurement of the crude polyphenoloxidase revealed that the polyphenoloxidase activity peak had a molecular weight of 60,000. In addition, MW-Marker (HPLC) of Oriental Industries, Ltd. was used as the molecular weight marker protein. Example 14: Cultivation and rough purification in a 5-liter flask

1% glucose and _{0.2% NH A C l, 1.34} % N a 9 HP_〇 _{_{4 · 1 2 H 2 0,}} 0.3% KH 2 P 0 4, 0.1% N a C 1, 0.2% peptone, 0.05% Mg S 0 _{_{4 · 7 H 9 0, 0.02mM}} C u S_〇 5 Li Tsu evening Ichiyo flask 2 device including the medium 1 Li Tsuta of ₄ to p H by adding 2 N-N a OH which was 8 Was inoculated with Myrothecium roridum (Myrothecium roridum) SD3002 (Accession No. FERM P-15255), and shaking culture was performed at 28 ° C for 3 days. After the cultivation, 1.6 liters of the culture broth was removed by centrifugation at 4 ° C.

Next, a portion of this culture pros was transferred to a minitan 'filter packet (CA

T. NO .: Concentrated as a fraction with a molecular weight of 10,000 or more using a minitan ultrafiltration system (Millipore) using PTGC0 P04 (Millipore). This was further subjected to DEAE-Ce1 lulofine A-800m column chromatography, and the eluted active fraction was reconcentrated again as a fraction with a molecular weight of 10,000 or more by the above-mentioned mini evening ultrafiltration system. did. Further, the reconcentrated solution was dialyzed against 200 ppm of NH ₄ HC 、 ₃ and then freeze-dried to obtain a crude product as a freeze-dried product. The freeze-dried product had a polyphenoloxidase activity of 5 U / mg. Example 15 Treatment of Contaminated Cloth with Detergent Containing Enzyme

25% by weight sodium linear alkylbenzene sulfonate (LAS), 5% by weight of polyoxyethylene lauryl ether, 15% by weight of sodium tripolyphosphate, 6% by weight of sodium silicate, 1% by weight of sodium carboxymethylcellulose, and 48% by weight of Na the index detergent 1 gram consisting ₂ S 0 _4, a material obtained by 0.01 g added pressure to the freeze-dried product of example 1 4, wherein the enzyme blended detergent, and enzyme Motohi formulated detergents those without added lyophilisate did.

Further, a contaminated cloth was prepared by adding 0.2 ml of 100 ppm Evans Blue (Wako Pure Chemical Industries, Ltd.) to the center of a cotton white cloth (5 cm x 5 cm). Next, after adding 1 piece of contaminated cloth and 1 Om 1 of water to a 500-m 1-volume beaker, add 1 O mg of a detergent containing enzyme or a detergent not containing enzyme, and shake for 12 minutes to wash. Was done. The stained cloth after treatment is washed with water and air-dried, and the Y, y, and x values (meaning the same meaning as above) and are measured by a colorimeter (CB-200, manufactured by MINOLTA).

Z = (1-X-y) Y / y

The difference between the Z values before and after the treatment (ΔΖ value) was calculated by the method. Example 16: Mixing of an enzyme derived from Myrothecium roridum SD3002 with a conventional polyphenoloxidase

An equal volume of a 0.1 μ / m 1 solution of polyphenol oxidase (derived from Bigidoporus s zona lis, commercially available as a reagent) and a 0.2 U / m 1 solution of the polyphenol oxidase of the present invention were mixed. When the activity was measured at various pH values, the oxidation reaction proceeded rapidly over a wide range of pH values from pH 5 to 11 as shown in FIG. Example 17: Measurement of isoelectric point The lyophilized product described in Example 14 was measured for isoelectric point by isoelectric focusing using a rotophor system (BI0-RAD). Pharmamarite (pH 2.5-5) (obtained from Sigma) was used as a buffer.

As a result, Myrothecium roridum S D 3

The isoelectric point of polyphenoloxidase derived from 02 (Accession No. FERM P-15255) was 4.1 ± 0.5. In addition, a polyphenoloxidase derived from Mvrothecium verrucaria SD 3001 (accession number FERM P-14955) was measured for the freeze-dried product described in Example 6 by the same method. The isoelectric point was 4.5 ± 0.5. Industrial applicability

As described in detail above, the present invention provides a polyphenol oxidase having an optimal reaction pH on the side of the pH of 8 or more, and by using this, enzymatic oxidation in an alkaline PH region. As a result, the field of use of polyphenol oxidase, such as oxidation of polyphenol substances and coloring substances, washing and bleaching, will be expanded.

In addition, the method for producing polyphenoloxidase of the present invention enables efficient production of the polyphenoloxidase of the present invention.

The Myrothecium verrucaria SD3001 and Myrothecium'roridum SD3002 of the present invention are effective for the production of the polyphenol oxidase of the present invention. A high effect can be obtained even when used directly in the bleaching process.

In addition, by using the polyphenoloxidase of the present invention, treatment of an effective coloring substance, treatment of paper, pulp or fiber, bleaching treatment, washing treatment, or sterilization or inactivation treatment of microorganisms or viruses, etc. the method of

Three 00948

PCT / JP96 / 01594

Is provided,

Claims

Claims 1) A polyphenol oxidase having an optimal reaction pH on the side of the microorganism having a pH of 8 or more. 2) The polyphenol oxidase according to claim 1, which is derived from a bacterium belonging to the genus Myrothecium. 3) The polyphenoloxidase according to claim 1 or 2 having the following properties. (1) Action It oxidizes polyphenol. (2) Optimal reaction pHHPH has an optimal reaction pH around 8.5-9. (3) Optimum reaction temperature The optimum reaction temperature is about 60 ° C. (4) Molecular weight The molecular weight measured by gel filtration chromatography is about 62,000. (5) Isoelectric point measured by isoelectric focusing is 4.5 ± 0.5. 4) The polyphenol oxidase according to claim 1, 2 or 3, which can be obtained from Myrothecium verrucaria. 5) The polyphenoloxidase according to claim 1, 2 or 3, which can be obtained from Myrothecium verrucaria SD3001 (accession number FEEJI P-14955). 6) The polyphenoloxidase according to claim 1 or 2 having the following properties.

(1) Action Oxidizes polyphenols.

(2) Optimal reaction pH

It has an optimal reaction pH around pH9.

(3) Optimal reaction temperature

It has an optimum reaction temperature around 70 ° C.

(4) Molecular weight

Molecular weight of about 60,000 as determined by gel filtration chromatography 0

(5) The isoelectric point measured by isoelectric focusing is 4.1 ± 0.5.

7) The polyfunox oxidase according to claim 1, 2 or 6, which can be obtained from Myrothecium roridum.

8) The polyphenoloxidase according to claim 1, 2 or 6, which can be obtained from Myrothecium roridum SD3002 (accession number FERii P-15255).

9) A phenol compound, an aromatic compound having an alkoxyl group, and a halogenated phenol, wherein the polyphenol-roxidase according to any one of claims 1 to 8 is acted on. A method for treating a compound or an aromatic amine compound.

10) A method for treating a colored substance, which comprises applying the polyphenol oxidase according to any one of claims 1 to 8.

1 1) A method for treating a microorganism or virus, which comprises causing the polyphenol oxidase according to any one of claims 1 to 8 to act.

12) Paper, pulp or fiber, which is made to act on the polyphenol-oxidase according to any one of claims 1 to 8. How to treat fibers.

13) The use of the polyphenol oxidase according to any one of claims 1 to 8, wherein the method is used together with a detergent, a detergent, or a surfactant.

14) The use of the polyfunoroxidase according to any one of claims 1 to 8, wherein the method is used together with a substance having a peroxidase action.

15) Any one of claims 1 to 8, wherein air, oxygen, ozone, hydrogen peroxide, hydrogen peroxide precursor, peracid precursor or peracid is used alone or in combination as an oxidizing agent. A method for using polyphenol oxidase, which is characterized in that it is used together with the polyphenol oxidase according to any of the above items.

16) The use of the polyphenol oxidase according to any one of claims 1 to 8, wherein the oxidase is used together with its substrate.

17) The method for producing polyphenol oxidase according to any one of claims 1 to 8, wherein a bacterium belonging to the genus Myrothecium is cultured.

18) The method for producing polyphenol oxidase according to claim 17, wherein the bacterium belonging to the genus Myrothecium is Myrothecium verrucaria.

19) The claim 17 wherein the bacterium belonging to the genus Myrothecium (My ^ theci_um) is Myrothecium verrucaria SD 3001 (Accession number: FERM P-14955) or a mutant thereof. The method for producing the polyphenol oxidase according to the above.

20) Myrothecium spp. 18. The method for producing a polyphenol oxidase according to claim 17, wherein the polyphenol oxidase is Myrothecium roridum.

21) The myrothecium genus Myrothecium roridum SD3002 (accession number FERM P-15255) or a mutant thereof is claimed in claim 17. A method for producing the polyphenoloxidase according to the above.

2 2) Myrothecium verrucaria ^ S D

3001 (accession number FEM P-14955).

2 3) Myrothecium roridum Sd302 (accession number FERM P-15255).

24) A detergent composition comprising the polyphenoloxidase according to any one of claims 1 to 8.